Acute kidney injury (AKI) disproportionately affects individuals in low-and middle-income countries (LMIC). However, LMIC—particularly countries in sub-Saharan Africa— are under-represented in global AKI research. A critical barrier in diagnosing AKI is access to reliable serum creatinine results. We evaluated the utility of a point-of-care test to measure creatinine and diagnose AKI in Ugandan children with malaria.Paired admission creatinine was assessed in 539 Ugandan children 6 months to 4 years of age hospitalized with severe malaria based on blood smear or rapid diagnostic test. Creatinine levels were measured using isotope dilution mass spectrometry (IDMS)-traceable methods. The reference creatinine was measured using the modified Jaffe method by a certified laboratory and the point-of-care testing was conducted using an i-STAT blood analyzer . AKI was defined and staged using the Kidney Disease: Improving Global Outcomes criteria.2 values of 0.95 and 0.93 respectively; however, the correlation was significantly lower in children with creatinine values <1mg/dL (R2 of 0.44 between the reference and adjusted and unadjusted i-STAT creatinine). The prevalence of AKI was 45.5% using the reference creatinine, and 27.1 and 32.3% using the unadjusted and adjusted point-of-care creatinine values, respectively. There was a step-wise increase in mortality across AKI stages, and all methods were strongly associated with mortality . AKI defined using the reference creatinine measure was the most sensitive to predict mortality with a sensitivity of 85.4% compared to 70.7 and 63.4% with the adjusted and unadjusted point-of-care creatinine values, respectively.The mean age of children was 2.1 years, and 21.6% of children were stunted. Mortality was 7.6% in-hospital. Over the entire range of measured creatinine values (<0.20mg/dL-8.4mg/dL), the correlation between the reference creatinine and adjusted and unadjusted point-of-care creatinine was high with RPoint-of-care assessment of creatinine in lean Ugandan children <4 years of age underestimated creatinine and AKI compared to the clinical reference. Additional studies are needed to evaluate other biomarkers of AKI in LMIC to ensure equitable access to AKI diagnostics globally. Acute kidney injury (AKI) is an important public health challenge, particularly in low-and middle-income countries (LMIC) where 85% of the estimated 13.3 million cases per year occur . AKI is Mortality in AKI in children in sub-Saharan Africa remains unacceptably high at 34% compared to a global average of 14% . FailureAKI is diagnosed based on increases in serum creatinine (SCr) or decreases in urine output . In pracAlthough children with severe malaria frequently present with multiple severe malaria complications and multi-organ dysfunction , 23, AKIWe evaluated the performance of the i-STAT1 handheld blood analyzer as a point-of-care tool to measure SCr and define AKI compared to a reference value obtained by a certified clinical laboratory. We present analyses comparing the correlation, bias, and precision of measures over the entire range of SCr values obtained. Further, as our population of interest is children <5 years of age with low estimated baseline SCr values, we conducted additional analyses focusing on the lower range of SCr values characteristic of lean pediatric populations. Using the SCr values from the different test methods, we assessed the prevalence and severity of AKI on admission and evaluated the ability of the different AKI definitions to predict mortality.Plasmodium falciparum histidine-rich protein-2 (HRP-2) or direct visualization of parasites by Giemsa microscopy, required hospitalization and met selected criteria for severe malaria . Exclusion criteria included known chronic illness requiring medical care, history of coma, head trauma, known developmental delay, cerebral palsy or prior hospitalization for malnutrition.Between 2014 and 2017, 600 children with a clinical definition of severe malaria were enrolled in the study. Children were eligible if they were between 6 months to 4 years of age, had diagnostic evidence of malaria with either a positive rapid diagnostic test for 2, PO2, TCO2, HCO3, base excess, sO2) on lithium heparin whole blood . Blood was tested immediately upon collection by trained nurses and medical officers following the manufacturer's recommendations. The i-STAT system is designed for in vitro quantification of SCr in arterial, venous, or capillary whole blood using 95uL of sample and provides results within two minutes with a reportable range for SCr of 0.20-20.0 mg/dL. SCr was measured using an enzymatic assay with amperometric detection on a platinum electrode. As the partial pressure of carbon dioxide (PCO2) can affect measurement of SCr by i-STAT, values were adjusted based on the PCO2 values as follows: for SCr values below 2 mg/dL and PCO2 values above 40 mmHg, [Cr]corrected = [Cr]i-STAT X {1 - (0.069 X [(PCO2 -40)/10])}; and for SCr values above 2 mg/dL, [Cr]corrected = [Cr]i-STAT X {1 - (0.037 X [(40 - PCO2)/10])} )} .Serum samples were shipped to the United States of America on dry ice and SCr was measured using a Beckman Coulter AU5822 chemistry analyzer using the modified Jaffe colorimetric method  at the Indiana University Pathology Laboratory. The limit of detection of the assay is 0.20 mg/dL, and the coefficient of variation of the assay is 4.1% at 0.80 mg/dL and 2.1% at 5.5 mg/dL.Both methods of SCr measurement were traceable to an isotope dilution mass spectrometry (IDMS) reference method using the U.S. National Institute of Standards and Technology (NIST) Standard Reference material SRM967. Samples below the limit of detection were assigned a value of 0.19 mg/dL.2 (height-independent), and Q=0.0270*age + 0.2329 [Acute kidney injury was defined using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria  based on+ 0.2329 . This me+ 0.2329 . A singlp value <0.05 was considered significant. The ability of AKI to discriminate between children who survived and children who died was assessed using non-parametric receiver operating characteristic (ROC) curve analysis using the method of Delong et al. [Data were analyzed using STATA v14.0 (StataCorp) and GraphPad Prism v7.03. Differences in SCr measures by test methodology were evaluated using a number of statistical tests. Concentrations of SCr were compared using Pearson’s correlation. The Bland-Altman method  was usedg et al. , and preThe funders had no role in the study design, analysis, or decision to publish.Of the 600 enrolled in the study, 539 children had paired SCr measures using the point-of-care i-STAT test and by the reference laboratory and are included in this analysis. The mean (SD) age of the 539 children enrolled in this study was 2.1 (0.9) years, with 42.9% of children female. The mean anthropometric z scores were -1.1 (1.1), -1.1 (1.3), and -0.7 (1.1) for weight-for-age, height-for-age and weight-for-height respectively. Undernutrition was common, with 116 (21.6%) of children stunted on enrollment. Overall, 11 children were living with HIV-1 (2.1%), and 21 (4.0%) children had a positive blood culture for bacteremia. Although all children enrolled were diagnosed with severe malaria based on a positive blood smear or a positive rapid diagnostic test for HRP-2, only 368 (68.3%) of children included in this analysis were smear-positive for malaria. A total of 41 children died during hospitalization (7.6%). The study flow is shown in Fig. PCO2) to the reference SCr from a clinical laboratory. The mean (SD) of the reference SCr value was 0.49 (0.56), and the unadjusted and adjusted i-STAT SCr values were 0.44 (0.63) and 0.47 (0.60), respectively. The values ranged from 0.19 mg/dL as the lowest value across all assays to the highest value of 7.3 mg/dL for the reference SCr, 8.4 mg/dL for the unadjusted i-STAT SCr, and 7.7 mg/dL for the adjusted i-STAT SCr. Overall, the correlation between the reference and point-of-care methods was high over the entire range of the assay with R2 values of 0.95 and 0.93 for the unadjusted and adjusted SCr values, respectively , the correlation was significantly lower with an R2 value of 0.44 between the reference SCr and both the unadjusted and adjusted point-of-care SCr measures . These findings were consistent across AKI definitions and AKI stages irrespective of the method to measure SCr Fig. , and the2 that is recognized to affect test performance. While the correlation between the two assays was high over the whole range of reported values (0.19-8.4 mg/dL), there was only moderate correlation across the range of lower SCr values typically measured in young children. When AKI was defined based on the unadjusted point-of-care SCr compared to the clinical reference, 99 (40%) of AKI episodes were missed (99/245). Although AKI was strongly associated with mortality across all AKI definitions with a step-wise increase in mortality across AKI stages, the relationship between AKI and mortality was strongest in children with SCr measured by the certified clinical laboratory.AKI is an under-recognized clinical complication associated with significant pediatric mortality globally. In the present study, we evaluated the ability of the i-STAT handheld point-of-care blood analyzer to measure SCr compared to a clinical reference using the modified Jaffe reaction. Compared to the certified clinical laboratory, the point-of-care test measured lower SCr values even after adjustment for the partial pressure of COThe reference method employed in this study was measuring SCr using the modified Jaffe reaction. Although the modified Jaffe reaction is recognized as traceable to the international standard, it is susceptible to interference through pseudo-chromogens resulting in SCr overestimation. Enzymatic reactions, like the ones used by the handheld i-STAT device, are generally considered more sensitive and specific than the Jaffe reaction. However, enzymatic SCr tests are considerably more expensive (estimated at $2.00 per test rather than $0.30) than the modified Jaffe reaction . Thus, tPCO2, the requirement for an additional cartridge to obtain the PCO2 measure makes it considerably more expensive to obtain adjusted values and impractical in a clinical setting.Although i-STAT SCr correlated strongly with the reference measure across the entire range of the assay, the correlation was only moderate in children with a SCr value <1 mg/dL. As this was a relatively young pediatric population and 21.5% of children were stunted according to WHO standards, the estimated baseline SCr was low across the population, so slight fluctuations in SCr had a relatively large impact on the estimated prevalence of AKI, reflecting limitations in SCr-based AKI definitions. Correspondingly, the AKI definitions differed in their ability to predict mortality. The unadjusted i-STAT SCr measure had a sensitivity of 63.4% to predict mortality compared to 85.4% using the reference method to define AKI. Although the sensitivity of the assay was better when adjusting for the Although the i-STAT had lower sensitivity to define AKI in this pediatric population, it remained strongly associated with mortality , and was able to correctly identify 41/50 (82%) of children with Stage 3 AKI where mortality was 32.0%. Thus, i-STAT remains a valuable point-of-care tool to identify children with AKI at increased risk of death in settings where laboratory assessment of SCr is unavailable. The i-STAT system would likely have better performance in adult populations where baseline SCr values are considerably higher, and identifying a 50% change in SCr from baseline would be less affected by assay imprecision. Most of the discrepancies in AKI in the present study were in defining Stage 1 AKI, which represented an average increase in SCr from an estimated baseline from 0.26mg/dL to 0.44 mg/dL.In a global survey published in 2017 to assess the current state of kidney care, only 12% of countries in Africa reported that they usually have the services to measure SCr with eGFR reporting at the primary care level, while 39% reported they never have the services available . FurtherAlthough it is not specific to AKI, blood urea nitrogen (BUN) accumulates when kidney function decreases. In Ugandan children with severe malaria, BUN is strongly correlated with AKI and outperforms serum SCr in predicting mortality . The prooC during hot seasons. Further, as i-STAT machines are not widely available in sub-Saharan Africa, it can be expensive and logistically challenging to maintain a supply of cartridges and controls that are not expired. Shelf-life is an important consideration when assessing the cost and performance of different platforms, and the ability to operate without a need for electricity in sites where there are frequent interruptions to grid electricity. A recent review assessed the strengths and limitations of existing point-of-care platforms for use in LMIC [A handheld point-of-care device that could be employed across all levels of health care would present an opportunity to substantially improve the recognition and management of AKI in resource-limited settings. In order for devices to be useful, they must be able to measure kidney function accurately, affordably, and should be amenable for use in remote tropical settings where heat and humidity can affect test performance . For exa in LMIC . All tes in LMIC . WithoutWhile a diagnosis of AKI currently requires a measure of SCr or assessment of urine output, other measures of impaired kidney function may have utility in identifying children with kidney dysfunction in LMIC. Additional studies are needed to evaluate the performance of existing AKI biomarkers (e.g., urine neutrophil gelatinase-associated lipocalin –44, CystIn this study, we found that the i-STAT measurement of SCr in a population of lean Ugandan children underestimated SCr compared to a clinical reference measure and led to the under-diagnosis of AKI. AKI was strongly associated with mortality irrespective of test methodology. The development of point-of-care approaches to measure SCr with good precision at lower SCr concentrations would be beneficial for pediatric populations. More importantly, there is an urgent need for AKI diagnostics that are temperature stable, affordable, and can be equitably accessed by patients in LMIC.


We report on a patient with an intracerebral hemorrhage (ICH), who showed delayed development of aphasia, which was demonstrated via follow up diffusion tensor tractography (DTT) to be related to neural degeneration of the arcuate fasciculus (AF).A 51-year-old, right-handed male presented with right hemiparesis, which occurred at the onset of a spontaneous ICH in the left corona radiata and basal ganglia. Brain magnetic resonance images showed a hematoma in the left subcortical area at one month after onset and hemosiderin deposits in the left subcortical area at nine years after onset. At four weeks after onset, he exhibited severe aphasia, and Western Aphasia Battery (WAB) testing revealed an aphasia quotient in the 39.6 percentile (%ile). However, his aphasia improved to nearly a normal state, and at three months after onset, his aphasia quotient was in the 90.5 %ile. At approximately eight years after onset, he began to show aphasia, and his aphasia increased slowly with time resulting in a WAB aphasia quotient in the 12.5 %ile at nine years after onset. The integrity of the left AF over the hematoma was preserved on 1-month post-onset DTT. However, the middle portion of the left AF in the middle of the hemosiderin deposits showed discontinuation on 9-year post-onset DTT. The fractional anisotropy value of the left AF was higher on the 9-year post-onset DTT (0.48) than that on the 1-month post-onset DTT (0.35), whereas the mean diffusivity value was lower on the 9-year post-onset DTT (0.10) than that on the 1-month post-onset DTT (0.32). The fiber number of the left AF was decreased to 175 on the 9-year post-onset DTT from 239 on the 1-month post-onset DTT.We report on a patient with ICH who showed delayed development of aphasia, which appeared to be related to degeneration of the AF in the dominant hemisphere. Our results suggest that DTT would be useful in ruling out neural degeneration of the AF. Stroke is often accompanied by neural degeneration –3. SeverRegarding intracerebral hemorrhage (ICH), the possibility of neural degeneration due to chemical injury by hematomal material has been suggested –9. SinceIn the current study, we report on a patient with ICH who showed delayed development of aphasia due to neural degeneration of the arcuate fasciculus (AF), which was demonstrated by performing follow up diffusion tensor tractography (DTT).A 51-year-old, right-handed male presented with right hemiparesis, which presented at the onset of a spontaneous ICH in the left corona radiata and basal ganglia . Brain m2; number of excitations = 1; and slice thickness of 2.3 mm with no gap (acquired voxel size 1.25 mm × 1.25 mm × 2.5 mm) [o.The DTI was performed twice (at one month and nine years after ICH onset). On both occasions, a sensitivity-encoding head coil on a 1.5-T Philips Gyroscan Intera scanner  with single-shot echo-planar imaging and navigator echo was used for the acquisition of DTI data.67 contiguous slices were obtained parallel to the anterior commissure–posterior commissure line for each of the 32 non-collinear diffusion-sensitizing gradients . DTI par 2.5 mm) . By usin 2.5 mm) . Image d 2.5 mm) . For ana 2.5 mm) . The fibThe integrity of the left AF over the hematoma was observed to be preserved on the 1-month post-onset DTT  at ICH onset; however, his aphasia had recovered to a nearly normal range (aphasia quotient: 90.5 %ile) at three months after onset. The integrity of the left AF just above the hematoma in the dominant hemisphere was shown to be preserved on the 1-month post-onset DTT (Fig. In conclusion, we report on a patient with ICH who showed delayed development of aphasia that may be related to degeneration of the AF in the dominant hemisphere. Our results suggest that DTT can be useful in ruling out neural degeneration of the AF. To the best of our knowledge, this is the first study to report on delayed neural degeneration of the AF nine years after the onset in a patient with ICH –9. Howev


E. coli K12, the vaccine construct was cloned into pET28a (+) vector.As the new pandemic created by COVID-19 virus created the need of rapid acquisition of a suitable vaccine against SARS-CoV-2 to develop Immunity and to reduce the mortality, the aim of this study was to identify SARS-CoV-2 S protein and N antigenic epitopes by using immunoinformatic methods to design a vaccine against SARS-CoV-2, for which S and N protein-dependent epitopes are predicted. B cell, CTL and HTL were determined based on antigenicity, allergenicity and toxicity that were non-allergenic, non-toxic, and antigenic and were selected for the design of a multi-epitope vaccine structure. Then, in order to increase the safety of Hbd-3 and Hbd-2 as adjuvants, they were connected to the N and C terminals of the vaccine construct, respectively, with a linker. The three-dimensional structure of the structure was predicted and optimized, and its quality was evaluated. The vaccine construct was ligated to MHCI. Finally, after optimizing the codon to increase expression in Epitopes which were used in our survey were based on non-allergenic, non-toxic and antigenic. Therefore, 543-amino-acid-long multi-epitope vaccine formation was invented through linking 9 cytotoxic CTL, 5 HTL and 14 B cell epitopes with appropriate adjuvants and connectors that can control the SARS coronavirus 2 infection and could be more assessed in medical scientific researches.We believe that the proposed multi-epitope vaccine can effectively evoke an immune response toward SARS-CoV-2.The online version contains supplementary material available at 10.1186/s43042-022-00224-w. Since the advent of SARS-CoV in 2002 and its spread to 32 countries, the world has experienced the outbreak of MERS-CoV and now nCoV 2019 . Their gThe use of epitope vaccines using immunogenic epitopes specific to CD8+ and CD4+ cells and stimulating the immune system against these epitopes simultaneously and completely specifically are among the methods that have been considered in this regard. Conventional methods for producing vaccines are time-consuming and expensive . The immThe aim of this study was to evaluate T-cell- and B-cell-dependent epitopes derived from SARS-CoV-2 S and N antigens for the design and development of a multi-epitope vaccine based on the analysis of immunoinformatic tools.https://ww.ncbi.nlm.nih.gov/) and also Human β-defensin-2 (PDB ID: 1FD3) and 3 (PDB ID: 1KJ6) from PDB database (https://www.rcsb.org).The FASTA sequences of S protein (YP_009724390.1) and N protein (YP_009724397.2) SARA-COV-2 were retrieved from the NCBI GenBank database (https://www.iedb.org/) and ABCPred (http://crdd.osdd.net/raghava/abcpred/ABC_submission.html). ABCPred ranks epitopes by using ANN scores, according to the score obtained and above the threshold (0.5); thus, it is more probable for a sequence to be an epitope with a higher score. For predicting linear epitopes with ABCPred server, 16 mer length of epitopes with default threshold (0.51) and for IEDB server with BepiPred linear epitope prediction method which predicts the location of B cell linear epitopes using a combination of a hidden Markov model and an orientation degree method were selected by default [An antigen must be able to elicit both the B and T cell immune responses in order to be a suitable candidate for the vaccine. Therefore, for predicting B cell linear epitopes two servers were used, IEDB ( default . From mahttps://webs.iiitd.edu.in/raghava/nhlapred.comp.html). 0.18 epitopes were selected with the highest score of alleles and with the highest frequency of Iranian population (HLA * A02:01) and (HLA * B35:01) according to the server  for analyzing the next ones. HTL epitopes for DRB1 * 0101, DRB1 * 1101 and DRB1 * 1501 alleles  from NetMHCIIpan 4.0 server (http://www.cbs.dtu.dk/services/NetMHC) were determined [In order to predict CTL epitopes, ComPred method  was used along with default cutoff score (0.5) of nHLAPred server (termined . The defhttps://www.ddg-pharmfac.net/AllerTOP/index.html) were reviewed to ensure the ability of selected epitopes to induce an immune response with the VaxiJen v. 2.0 server (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) and were examined with a threshold of 0.4. This is because toxic epitopes can compromise the structure of the vaccine and should be removed. ToxinPred server (https://webs.iiitd.edu.in/raghava/toxinpred/design.php) with SVM method and default server parameters was used to determine toxic epitopes. Finally, antigen, non-allergenic and non-toxic epitopes were selected as possible epitopes for CTL and HTL B cell.Since the components of the vaccine must be capable of allergic reactions, the selected epitopes for B cell, CTL and HTL with the AllerTOP server v. 2.0  [http://imed.med.ucm.es/Tools/antigenic.pl) [http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) [http://scratch.proteomics.ics.uci.edu/) [https://web.expasy.org/protparam) [Allergenicity assessment has the ability to predict the structure of the vaccine in causing allergies and allergic reactions. Accordingly, the Allergen FP 1.0 server (rgenFP/)  was usedenic.pl)  and Vaxien.html)  was examci.edu/) . The Prootparam)  was usedhttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) and the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) were used to identify the second structure of the vaccine construct, and the phyre2 server (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index), RaptorX server (http://raptorx.uchicago.edu/ContactMap/) and I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER//) were used to predict the third structure of the vaccine.SOPMA server . ProSA, ERRAT and Ramachandran servers in the software (https://servicesn.mbi.ucla.edu/PROCHECK/) were used to validate the optimized 3D structure [To identify and correct the errors of the selected 3D model, the 3D structure was optimized by using the GalaxyRefine server (tructure –39.https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/) [http://www.jcat.de/) [E. coli K12 to optimize the codon structure of the multi-epitope vaccine. Finally, the construct sequence of the optimized multi-epitope vaccine was cloned into the pET28a (+) vector using The SnapGene program. Virtual agarose gel simulation was used to virtualize the clone.The Backtranseq server (ranseq/)  was usedcat.de/)  was usedhttps://cluspro.org) [Server ClusPro 2.0 (pro.org)  was usedpro.org) . The besMolecular dynamics is a computational method that was conducted to demonstrate the behavior of molecules and to evaluate the stability of protein–protein complexes . In thisTo evaluate the immunogenicity of the ultimate vaccine, in silico immune simulations were performed by utilizing the C-ImmSim server. This immune trigger applies a position-specific scoring matrix (PSSM) and machine learning methods in order to estimate epitope prediction and immune interactivities, respectively .Clinically, the minimum period of time suggested between two doses of vaccines is 1 month . Immune The overlap results of the predicted linear B cell epitopes which were found by IEDB and ABCPred servers for proteins S and N are shown in Table The prediction results of CTL epitopes (9 mer) with nHLAPred server Table  and HTL The accuracy of the epitopes predicted for B cell, CTL and HTL, in terms of allergenicity, antigenicity and toxicity, was examined. In order to design the vaccine structure, both non-allergenic and non-toxic epitopes that had antigenic potency were selected. Finally, 9 CTL epitopes (5 epitopes for N protein and 4 epitopes for S protein), 5 HTL epitopes . Based on the results of the SOLpro server, the designed vaccine structure was predicted soluble with a probability of 0.9, which ensures easy access to the host. Also designed vaccine structures were predicted to be non-toxic, non-allergenic and antigenic. The result of its antigenicity according to VaxiJen and ANTIGENpro servers is 0.5 and 0.9, respectively.Examination of the stereochemical properties of the designed structure using ProtParam program showed that the molecular weight of the vaccine structure is 59038.88 daltons with an isoelectric point of 10.06 which shows the basic nature of the designed vaccine structure. The total number of negatively charged amino acids  is 39, and the total number of positively charged amino acids  is 104. The aliphatic index is 57.24 and the instability of the designed structure was reported to be 38.96, which indicates the stability of the vaccine structure designed in the host. GRAVY index was − 0.7, and it was reported that the negative of this index indicates that the vaccine structure is hydrophilic, so it could interact well with water molecules. The half-life of this vaccine construct was predicted to be 30 h in mammals (in vivo), more than 20 h in yeast (in vivo) and more than 10 h in The second structure of the protein using the PSIPRED program is shown in Fig. Structural validation is a procedure to recognize potential flaws in the estimated tertiary structure . The oveIn order to evaluate the cloning and expression of the vaccine construct in the expression vector, the inverse translation of the vaccine construct sequence was received by the Backtranseq server and its codon was optimized by the JCAT server. The JCAT server evaluates the sequence to optimize the codon and reports the codon compatibility index (CIA) and GC content of the sequence. According to the results, the codon compatibility index (1.0), which was in the optimal range (0.8–1.0), was calculated. A high CIA value indicates high gene expression. Also, GC sequence content 51.93%) was in the desired range (30–70%). These results may indicate high expression of the vaccine construct in the bacterial system. Finally, after adding the BamHI and XhoI restriction enzymes to the sequence, the optimized codon sequence was cloned using the SnapGene program in the pET28a (+) vector Fig. . The Sna1.93% wasDocking of the designed vaccine construct as a ligand with HLA-A02;01 (PDB ID: 3TO2) as the receptor was performed by the ClusPro 2.0 server. This server predicts 30 complexes and classifies them based on the amount of energy. Among the predicted models, model 4, which had the lowest energy weighted score of − 1158.9, has been selected as the best model for vaccine interaction with HLA-A02:01 Fig. . Additio−08. The covariance matrix between the pairs of residues is displayed in Fig. To assess the firmness and physical motions of the created vaccine composition—HLA-A02:01 docked compound. Molecular dynamics simulation was performed through the iMOD server . The maiThe immunogenic profile of the designed vaccine candidates was attained from C-IMMSIM server. Simulation outcomes depicted that high concentrations of IgM were recognized at the primary response. In both secondary and tertiary responses, the usual elevated levels of immunoglobulin activities  were noticeable with associated antigen depletion Fig. A. The elN234ITRFQTLLALHR, FSN61VTWFHAIHVSGT, TESIVRFPN331ITNLCP) contain glycosylation sites, which should have a minimum influence on antigen recognition. If these glycosylation sites hinder the diagnostic presentation, an extra deglycosylation step with N-glycanase should be useful for the test samples, which is a simple and useful technique for deglycosylation [Today, as coronaviruses appear periodically and unpredictably and they are spreading rapidly, they are causing serious infectious diseases; they have become a constant threat to human health. This is especially true when there is no vaccine or approved drug to treat coronavirus infection . Many stsylation . Owing tsylation . In thissylation . Accordisylation .E. coli. Based on the results, the structure of the designed vaccine solution was predicted to ensure easy access to the host. Also, according to the predicted results, the structure of the designed vaccine is antigen, non-toxic and non-allergenic. The quality of the three-dimensional structure of the designed vaccine structure increased dramatically after optimization, so that all amino acids in the desired and allowed areas (100%), according to Ramachandran map, reported that it shows the appropriate quality of the three-dimensional structure of the designed vaccine structure. Various tools were used to determine possible errors and the quality of the three-dimensional structure of the designed vaccine structure. Z-score (− 9.22) and ERRAT quality factor  showed that the structure of the designed vaccine is appropriate. Using ClusPro2.0 server, connection was made between the vaccine structure designed with HLA-A02:01—1158.9 of HLA-A02:01 was the lowest amount of energy in the total of the vaccine structure. Furthermore, the iMODS server was applied to evaluate the constructional steadiness and atomic-level motions of docked complex (designed vaccine construct—HLA-A02:01). It showed that docked proteins have minor deformation for each residue and with establishing our estimation of eigenvalues for 3.23e−08, which display the validity of our in silico predicted vaccine. Because all codons that are synonymous in a codon family do not use the same rate of expression of heterogeneous proteins in Escherichia coli, codon optimization in production of eukaryotic proteins is necessary in prokaryotic hosts [E. coli K12, and according to the results, both codon compatibility index (1.0) and GC percentage (51.93%) were calculated; this reveals a high probability of protein expression in bacteria. In addition, the immune simulation of the designed vaccine structure showed hopeful results regarding both humoral and cellular immune reaction. The results of bioinformatics evaluation of the designed vaccine construct indicated that this vaccine candidate may be highly potent against SARS-CoV-2, but in vitro and in vivo studies are needed for clinical confirmation.The molecular weight of the designed vaccine was 59,038.88 daltons (approximately 59 kDa), which makes it an acceptable vaccine. Because proteins with a molecular weight of less than 110 kDa are considered as more suitable targets for vaccine production . The isoic hosts ; therefoIn silico vaccine formation being efficient is substantially important, and it strongly focused on the multi-epitope peptides of the vaccine. In this study, using bioinformatics analyses, suitable epitopes of S and N proteins were selected and analyzed. Finally, a different multi-epitope vaccine with a span of 543aa against the 2019-nCov will be created.It consists of two adjuvants, with 14 B cell epitopes, 9 CTL epitopes and 5 HTL epitopes. It displays good antigenic features, immunological qualities and satisfactory physiochemical characteristics, non-allergenicity and non-toxicity. It is expected that the epitopes predicted in this study would be an efficient vaccine formation against COVID-19. However, the confirmation of the epitopes which were selected in this study as a vaccine candidate should be considered as laboratory studies.Additional file 1. Design of a multi-epitope-based peptide vaccine against the S and N proteins of SARS -COV-2 using Immunoinformatics Approach. .


In this work, we report the synthesis of 5-fluorouracil (5-FU)-imprinted microparticles and their application in prolonged drug delivery. The materials were synthesized using either ethylene glycol dimethacrylate (EGDMA) or trimethylolpropane trimethacrylate (TRIM) cross-linkers. For both types of polymers, methacrylic acid was used as a functional monomer, whereas 2-hydroxyethyl methacrylate was applied to increase the final materials’ hydrophilicity. Adsorption isotherms and adsorption kinetics were investigated to characterize the interactions that occur between the materials and 5-FU. The microparticles synthesized using the TRIM cross-linker showed higher adsorption properties towards 5-FU than those with EGDMA. The release kinetics was highly dependent upon the cross-linker and pH of the release medium. The highest cumulative release was obtained for TRIM-based microparticles at pH 7.4. The IC Drug delivery systems (DDS) are sophisticated technologies that allow targeted delivery of particular pharmaceutical or controlled therapeutic agents’ release. DDS application is essential for therapeutic agents limited due to their low solubility, drug administration issues, or very fast metabolism. Moreover, the application of DDS allows to optimize drug efficiency and improve administration, while lowering the possibility of causing adverse side effects. As a result, the development of new DDS occurs in parallel with creating new therapeutic agents. Whenever a new drug has a limited therapeutic effect in a free form, the combination of drugs and DDS may overcome this problem. The design, synthesis, and production of new substances and materials that can be applied to prepare new DDS have become a major topic of many research groups. Their goal is to develop a DDS which would allow drug administration to a specific site with a known quantity and for a precise amount of time. It is, of course, necessary to produce DDS that, as well as their metabolites, show no toxicity and are easily removed from the human body ,2,3.Various materials have been used to generate DDS. The most frequently used are lipids ,5, chitoMIPs are synthesized by copolymerizing a cross-linker mixed with a complex formed by functional monomers and template molecules. The cross-linker initiates the formation of the bulk polymer structure and entrapment of the template molecules inside, whereas functional monomers form stable, non-covalent interactions with template molecules. Finally, it results in the synthesis of MIPs selective towards the template used, which can be reversibly bound by cavities formed during the reaction. As a result, they have found many applications in fields such as solid-phase extraction (SPE) ,28,29, dMany researchers find the application of MIPs in drug delivery particularly interesting due to the possibility of producing materials that alter their interaction strength with drug molecules influenced by a specific change in the environment. MIPs possess an enhanced affinity to the drug template, which increases the residence time of the drug. Moreover, MIPs’ properties allow for reaching high drug loading, and the fact that the drug is entrapped within the polymeric network results in its higher stability and durability against harsh conditions. MIPs can be synthesized as smart materials that can be effectively used to produce DDS, considering that they can be programmed to release therapeutic agents as a response to defined stimuli . MIPs caMIPs are synthesized as monoliths that are ground to obtain particles of desired dimensions for numerous applications. Although this process is frequently used in analytical applications such as SPE, the development of DDS requires more rigorous control over polymer particles’ size. New synthetic methods that allow obtaining MIPs as uniform nanoparticles or microparticles have been developed to solve this problem. As synthesized materials possess a significantly increased surface area, more cavities are closer to the surface, making them easily accessible. The additional benefit of small particle sizes is their ability to form stable dispersions in various solvents, which increases their attractiveness as potential materials to be used as DDS ,40,41.In this work, we report the synthesis and characterization of MIPs microparticles imprinted with 5-fluorouracil (5-FU). 5-FU is a cytotoxic drug with a broad activity spectrum against numerous tumors. It undergoes fast metabolism in the human body, and thus a therapy consisting of high doses is required, which can cause severe toxic effects in many patients . As a reEthylene glycol dimethacrylate, methacrylic acid, trimethylolpropane trimethacrylate, 2-hydroxyethyl methacrylate, 5-fluorouracil, 2,2′-azobisisobutyronitrile solution , and all solvents (HPLC grade) were obtained from Sigma-Aldrich .−1) at a heating rate of 10 °C min−1. Scanning electron microscopy (SEM) images were recorded using a Scanning Electron Microscope SU3500 . UV-Vis absorption spectra were obtained using a 8453  spectrophotometer. The solutions’ pH values were controlled with an CP-505 pH meter .The FTIR measurements were performed using a IFS 66/s spectrometer . To obtain spectra, 1.5 mg of each material was mixed with ca. 200 mg of KBr, and the resulting powder was converted into tablets used in FTIR measurements. Thermogravimetric (TG) analysis was performed using a Setsys 1200  apparatus. The analysis was performed in an air stream (50 mL minEGDMA) were finally dried under a vacuum.MIPs created using EGDMA cross-linker were synthesized as follows. The pre-polymerization mixture consisting of 5-FU (0.9 mmol), MAA (1.8 mmol), HEMA (0.9 mmol), and methanol (90 mL) was prepared in a glass pressure tube. The mixture was degassed for 30 min using an ultrasound bath and purging with inert gas (nitrogen). Subsequently, EGDMA (8 mmol) and AIBN solutions (1 mL) were added, and the tube was degassed for an additional 10 min. Afterward, the tube was sealed and placed at 60 °C for 18 h. As-synthesized microparticles were filtered off and dried under a vacuum. The resulting drug-loaded MIPs were placed in a dialysis tubing to remove template molecules and were dialyzed against acidified methanol (9:1 methanol/acetic acid). This process was continued until 5-FU was no longer detected in a dialysis solution. The drug-unloaded MIPs cross-linked with EGDMA , MAA (4 mmol), HEMA (2 mmol), and methanol (80 mL). After degassing and sonication, TRIM (4 mmol) and AIBN solutions (1.4 mL) were added, and the tube was degassed for an additional 10 min. The remaining procedure is identical to EGDMA-based MIPs. Finally, the drug-unloaded MIPs cross-linked with TRIM were denoted as MIPEGDMA and NIPTRIM. The structures of all used monomers and templates are presented in The corresponding non-imprinted polymers (NIPs) were synthesized using analogous procedures but without adding template molecules. The obtained microparticles were denoted as NIP−1 was added to 10 mg of MIPs or NIPs. The obtained mixtures were equilibrated for 24 h at ambient conditions (25 °C). UV-vis absorption spectra were taken prior to and after adsorption solutions to establish the concentration of 5-FU. The amount of adsorbed 5-FU  was calculated as follows:C0 is the initial concentration of 5-FU solution (mg mL−1), eqC is the equilibrium concentration of 5-FU solution (mg mL−1), m is the polymer (MIPs/NIPs) mass (g), and V is the volume of 5-FU solution (mL). The experiments were repeated three times, and mean values were used for calculation.Adsorption isotherms were established using batch experiments, in which 10 mL of methanolic 5-FU solution at concentrations ranging from 0.39 to 50 mg L−1. The solution was stirred at ambient conditions, and the concentration of 5-FU was measured at defined time intervals by UV-vis absorption spectra. The tq value (mg g−1), which represents the amount of adsorbed 5-FU, was calculated as follows:tC is the concentration of 5-FU after time t (h).The adsorption kinetics studies were conducted when 50 mg of either MIPs or NIPs were placed in 50 mL of 5-FU solution in methanol with an initial concentration of 10 mg LThe dissolution method was used to conduct the in vitro release studies. The experiments were conducted by dispersing 5-FU-loaded MIPs (20 mg) in buffer solutions (10 mL) at pH 2.2, 5.0, and 7.4. The solutions were stirred continuously for seven days at 37 °C, and at specified time intervals, the samples were collected, centrifuged, and their UV-vis absorption spectra were recorded. The obtained data allowed to calculate the amount of drug releases from studied MIPs. The experiments were repeated three times, and the obtained mean values were used for subsequent calculations.tF is the amount of 5-FU released at a specified time “t”, F0 is the initial amount of 5-FU in MIPs structure, k0, k1, kH, kHC, and kKP are the release constants of corresponding equations, and n is the diffusion exponent.The results obtained for the 5-FU release were fitted using various models that characterize the mechanism of the release process. The following models were applied: zero-order (Equation (3)), first-order (Equation (4)), simplified Higuchi (Equation (5)), Hixson–Crowell (Equation (6)), and Korsmeyer–Peppas (Equation (7)). The following equations mathematically represent these models:The cytotoxicity assays were performed using the experimental procedure described in our previous research. Briefly, KB, HeLa, and MCF-7 cell lines were obtained from The European Collection of Cell Cultures (ECACC) supplied by Sigma-Aldrich . whereas A-549, U-87MG, and HDF cell lines were purchased from the American Type Cell Collection (ATCC) through LGC Standards. Approximately 0.1 mL of the diluted cell suspension  was added to every well of the microtiter plate. A partial monolayer was formed after 24 h, and the supernatant was washed out. Then, 100 μL of 6 different 5-FU concentrations  or number of MIPs that release the corresponding amount of 5-FU were added to the cells in microtiter plates. For NIPs blank experiments, the same mass of microparticles was used as for MIPs. Other experimental details were described in our previous work.p-value lower than 0.05 was considered as statistically significant.One-way ANOVA with the post-hoc Tukey HSD test was used to test statistical significance. A −1, respectively. These bands overlap with O-H stretching, and bending vibrations originated from hydroxyl groups of HEMA. The asymmetric stretching vibrations of CH2 groups can be observed at 2955 cm−1 for EGDMA-based microparticles and 2974 for TRIM-based microparticles. The stretching vibrations of C=O bonds are observed at 1730 cm−1 for EGDMA-based microparticles and 1734 cm−1 for TRIM-based microparticles. The symmetric and asymmetric C-O stretching vibrations of ester groups can be observed at 1258 and 1161 cm−1 for EGDMA-based microparticles, respectively. For TRIM-based microparticles, these signals are observed at 1268 and 1156 cm−1, respectively [EGDMA shows a band characteristic for 5-FU at 816 cm−1, whereas for drug-loaded MIPTRIM, these additional bands can be observed at 816, 553, and 471 cm−1.The synthesized imprinted microparticles and corresponding non-imprinted microparticles present similar bands in the IR spectra , which iectively ,47. DrugEGDMA present microparticles of 0.7–1.3 µm in diameter, whereas MIPEGDMA microparticles are much smaller, ranging between 300 and 600 nm. This result clearly indicates that the presence of 5-FU in the polymerization mixture results in the formation of smaller microparticles. The NIPTRIM microparticles are also smaller than those synthesized using EGDMA as a cross-linker, and their size ranges between 400 and 800 nm. Contrary to the previous observation, the presence of 5-FU in the polymerization mixture has not influenced the size of MIPTRIM microparticles. This result may indicate that the presence of three methacrylate groups in the cross-linker structure that undergo polymerization makes the process less prone to the presence of additional substances in the reaction mixture that can affect the reaction.SEM images of EGDMA- and TRIM-based microparticles are presented in EGDMA microparticles, an increased weight loss is observed in the initial decomposition step compared to drug-unloaded material. It probably results from the decomposition of 5-FU molecules. Compared to EGDMA-based microparticles, all TRIM-based microparticles are characterized by only one major decomposition step, that starts at around 300 °C and ends at around 475 °C, reflecting almost complete oxidation of the polymer material. For 5-FU-loaded MIPTRIM, only a slight increase in the weight loss of the initial decomposition step is observed compared with drug-unloaded material, which is connected with the decomposition of 5-FU molecules. The increase in the weight loss occurs within the 260–320 °C range, which is in accordance with the decomposition temperature of 5-FU reported in the literature [The TG results obtained for EGDMA-based microparticles are presented in terature .The adsorption process’s characterization is achieved by plotting adsorption isotherms for experimental data obtained during adsorption of 5-FU by the microparticles at the equilibrium state. K (L mg−1) is the binding equilibrium constant, mq (mg g−1) is the maximum amount of bonded 5-FU, eqC (mg L−1) is the equilibrium concentration of 5-FU, and eqq (mg g−1) is the amount of 5-FU adsorbed at the equilibrium concentration. K, qm, and R2 (correlation coefficients). The R2 values obtained for all synthesized microparticles are high (above 0.98), indicating that the Langmuir adsorption model nicely fits the experimental data. The confirmation of successful imprinting can be found in the high difference between the maximum adsorption capacity (mq) between MIPs and corresponding to them NIPs. For both MIPs–NIPs pairs, the mq value is around three times higher in favor of MIPs. There is also a significant difference in mq values between the corresponding materials synthesized using different cross-linkers. The calculated data clearly show that the maximum adsorption capacity values are higher for MIPTRIM and NIPTRIM than for MIPEGDMA and NIPEGDMA, respectively. As a result, it can be concluded that a TRIM cross-linker allows constructing a polymer network that has a higher affinity to 5-FU and allows to imprint it more effectively than a network synthesized using the EGDMA cross-linker.The following equation represents the Langmuir adsorption isotherm:Kf and n represent the Freundlich constants, eqC (mg L−1) is the equilibrium concentration of 5-FU, and eqq (mg g−1) is the amount of 5-FU adsorbed at the equilibrium concentration. Kf, 1/n, and R2 (correlation coefficients). The R2 values obtained for MIPEGDMA, NIPEGDMA, and NIPTRIM are higher than 0.97, suggesting that the experimental data can be fitted using the Freundlich adsorption model. On the other hand, the R2 values obtained for MIPTRIM are lower than 0.94, indicating that the Freundlich adsorption model should not be used to characterize the experimental results. The 1/n value calculated from the Freundlich adsorption model is considered a measure of adsorption intensity or surface heterogeneity. Basically, the closer the 1/n value to zero, the more heterogeneous the surface [n is higher than one, then adsorption is considered cooperative [n values obtained for all microparticles are similar and are in the 0.60–0.73 range. This result indicates that all materials’ heterogeneity is similar and the adsorption of 5-FU on them follows a similar mechanism.The following equations represent the Freundlich adsorption isotherm: surface . Moreoveperative . The 1/ntq versus t were obtained and are shown in k1 (h−1) is the pseudo-first-order rate constant, qe (mg g−1) is the amount of 5-FU adsorbed at the equilibrium concentration, and qt (mg g−1) is the amount of 5-FU adsorbed at time t (h). The k1 and R2 values are presented in 2 values range between 0.979 and 0.993, which means that this model could be applied to characterize the kinetics of the adsorption of 5-FU on synthesized microparticles. However, after comparing these results with much higher R2 values calculated for the pseudo-second-order kinetic model, it becomes clear that the pseudo-first-order kinetic model only partially fits the experimental data obtained for all microparticles.Adsorption kinetics can be established by finding a relationship between the adsorption capacity of examined materials and the contact time with the adsorbate solution. For all synthesized microparticles, the plots of k2 (g mg−1 h−1) is the pseudo-second-order rate constant. The k2 and R2 values are presented in 2 values calculated using this model are much higher than those obtained for the pseudo-second-order kinetic model and are in the 0.994–0.999 range. This result indicates that the pseudo-second-order kinetic model should be used to characterize the adsorption of 5-FU by synthesized microparticles. For both MIPs and NIPs, the k2 values are quite similar and are within a level of 0.52–1.73, indicating that the kinetics of 5-FU adsorption for all materials is similar.The following equation represents the pseudo-second-order kinetic model:p < 0.05). For EGDMA-based MIPs , the highest cumulative release (ca. 40%) was observed for pH 2.2, whereas a much lower release, equal to 20% and 12%, was observed for pH 5.0 and 7.4, respectively. This result clearly shows that the higher the pH, the lower the cumulative release. The observed behavior is probably caused by the higher protonation of carboxylic groups present in the polymer structure at lower pH values. This process disrupts interactions between carboxylic groups and drug molecules, leading to increased drug release. The release profiles obtained for the examined pH values are different depending on the buffer pH. For pH 5.0 and 7.4, a very high initial burst release is observed, which means that after around 10 h, the release almost entirely reaches the final cumulative release values. On the other hand, the initial burst release at pH 2.2 is much lower, and after that, a steady release is observed, which lasts around 50 h, after which a final cumulative release value is reached. For TRIM-based MIPs (drug loading 24.2 mg g−1), a completely different behavior is observed regarding the dependence of pH of the release medium on the cumulative 5-FU release. The highest cumulative release (ca. 86%) was observed for pH 7.4, whereas for pH 5.0 and 2.2, these values were lower, equal to 70% and 53%, respectively. This behavior, almost opposite to the results obtained for MIPEGDMA, is presumably caused by the much higher hydrophobicity of the TRIM-based polymer network than the one observed for EGDMA-based polymers. As 5-FU is sparingly soluble in water, it possesses a relatively high affinity towards the hydrophilic environment, and therefore the hydrophobic TRIM-based polymer network does not form strong interactions with 5-FU drug molecules. As a result, at pH 7.4, the drug release from MIPTRIM has the highest value because, at this pH value, the polymer network’s hydrophobicity is the highest. When the pH is lowered, the polymer’s structure becomes protonated, increasing its hydrophilicity, leading to a lower 5-FU release from its structure. Moreover, a rapid release at pH 2.2 can be observed, which is probably connected with breaking interactions occurring between MIPs cavities and 5-FU. As a result, during release, the amount of 5-FU maintained in the MIPTRIM structure is physisorbed primarily on its structure due to the polymer’s increased hydrophilicity. The higher hydrophobicity of the TRIM-based polymer network than the EGDMA-based network also explains the much higher cumulative percentage release of 5-FU for MIPTRIM than the one observed for MIPEGDMA.Three buffer solutions of pH 2.2 (simulated gastric fluid), pH 5.0 (simulated tumor interstitium of tumor cells) ,52, and EGDMA and MIPTRIM. The release profiles were fitted with zero-order, first-order, Higuchi, Hixson–Crowell, and Korsmeyer–Peppas release models. The values of corresponding release constants (k), correlation coefficients (R2), and the diffusion exponents (n) are presented in 2 values were calculated for the Korsmeyer–Peppas release model, which indicates that the experimental data is best fitted with this model. The only exception is the release of 5-FU from MIPTRIM at pH 5.0, for which a higher R2 value was obtained for the Higuchi model (R2 = 0.977) compared to the Korsmeyer–Peppas model (R2 = 0.956). However, the differences in the R2 values are not significant; therefore, it can be concluded that the experimental data is nicely fitted with both of these models. These results indicate that the release mechanism of 5-FU from both MIPs at all examined pH values is similar. Moreover, for all MIPs at all examined pH values, the diffusion exponent value in the Korsmeyer–Peppas model was lower than 0.45, indicating a Fickian diffusion-controlled release mechanism.Several mathematical models were used to fit the experimental data obtained from the 5-FU release from MIP50 values. Both drug-loaded MIPs present very high cytotoxicity against cancer cell lines, contrary to the result obtained for drug-unloaded MIPs. The results clearly show that drug-loaded MIPTRIM shows almost identical cytotoxicity against HeLa cancer cell lines as pure 5-FU. In contrast, its cytotoxicity against normal HDF cell lines is nearly three times lower than that measured for 5-FU. As a result, it can be concluded that MIPTRIM can be a good candidate as a DDS for 5-FU because it allows maintaining high cytotoxicity against HeLa cell lines while lowering the cytotoxicity against healthy cells.Cytotoxicity against U87 MG, HeLa, KB, A-549, and MCF-7 cancer cell lines, and HDF normal cell lines, was examined for drug-loaded and drug-unloaded MIPs and pure 5-FU. 50 values proved that drug-loaded MIPTRIM possesses high cytotoxic activity against cancer cell lines, while lowering the toxicity towards normal HDF cell lines. Therefore, it has been proven that the selection of a cross-linker has a significant impact on the final properties of microparticles and has to be considered during the design of materials used for drug delivery.We have shown that microparticles imprinted with 5-FU can be applied for the prolonged release of this drug. The microparticles synthesized using the TRIM cross-linker showed higher adsorption properties towards 5-FU than those synthesized using EGDMA. The experiments proved that prolonged drug release could last up to 50 h when an EGDMA cross-linker is used. The calculated highest cumulative release was highly dependent on the cross-linker applied during the synthesis. For EGDMA-based MIPs, the highest cumulative release was observed at pH 2.2, lower at pH 5.0, and the lowest at pH 7.4. Opposite results were obtained for TRIM-based MIPs, as the highest cumulative release was obtained for pH 7.4 and the lowest for pH 2.2. Moreover, the overall cumulative release was much higher for TRIM-based than EGDMA-based MIPs. The 5-FU release from all examined materials at all pH values was well fitted with the Korsmeyer–Peppas model. The IC


Studies increasingly show that positive psychological constructs affect the mental health of cancer patients. However, most scales that measure hope, resilience, optimism and self-efficacy have been developed based on general populations. The aim of our study was to develop a psychological capital (PsyCap) questionnaire for patients with cancer (PCQ-C) to gauge their mental state more accurately.The items for the scale were selected by comprehensive literature review and semi-structured interviews, and the relevant terms were screened by an expert panel. A pilot study was then conducted on 202 patients to reduce the item pool, and the reliability and validity of the scale were evaluated using 500 completed questionnaires. The test-retest reliability was then assessed using a subsample of 100 patients. Finally, the completed questionnaires of 229 patients with breast cancer were used to assess the criterion validity of the PCQ-C, including measures of depression and anxiety.r = − 0.631, P < 0.01) and anxiety .Item reduction and exploratory factory analysis resulted in 24 items for self-efficacy, hope, resilience and optimism, accounting for 56.72% of the variance. The Cronbach’s alpha for the scale was 0.886, and the test-retest reliability was 0.825. PsyCap showed a significant negative correlation with both depression (The PCQ-C can objectively evaluate PsyCap in cancer patients and exhibits good psychometric properties. Several state-like positive psychological constructs have been identified in the fields of positive psychology and psycho-oncology , 2. The Cancer is the leading cause of death globally, and according to World Health Organization (WHO) reports, it is the first or second leading cause of death among those younger than 70 years in most countries . Cancer However, the current evaluation scales are based on participants with depression and anxiety or the general population , 16. ForThe survey was administered to cancer patients across five cities in northeast China between September 2020 and February 2021. The inclusion criteria for patients were as follows: 1) cancer diagnosis, 2) awareness of the disease, 3) fluency in Chinese, and 4) 18 years or older. Patients with other severe diseases  were excluded. The participants were interviewed to obtain all relevant information. The study was approved by the Ethics Committee on Human Experimentation of China Medical University, and all participants provided written consent prior to the survey.Depression was evaluated using the Chinese version of the self-reported Patient Health Questionnaire-9 (PHQ-9)  over a pAnxiety was assessed using the Chinese version of the self-reported Generalized Anxiety Disorder-7 (GAD-7)  over twoThe preliminary questionnaire was developed on the basis of a literature review and semi-structured and in-depth interviews with 30 cancer patients. Following discussion with an expert panel of psycho-oncologists and grouping/excluding any similar or ambiguous items, a 64-item questionnaire was drafted. Each item was scored on the basis of a five-point Likert scale that varies from “1=very strongly disagree” to “5=very strongly agree”. A pilot survey was then carried out on 202 cancer patients to assess the validity and clarity of the questionnaire, and the number of items was reduced to 44.r < 0.1) were excluded, and the highly interrelated items (r > 0.7) were analysed further. The least clinically relevant item was excluded due to high association with the same underlying dimension theoretically [A total of 505 cancer patients were recruited on the basis of the inclusion and exclusion criteria, and their demographic and clinical data were collected. The intercorrelation between the items was calculated based on the data generated from the survey. Low interrelation items  was used to assess construct validity to identify the key components. The suitability of EFA was assessed by Bartlett’s test, Kaiser-Meyer-Olkin (KMO) measure and eigenvalue cut-off value > 1. The items with total correlation < 0.3, factor loading < 0.5 on one factor, communality < 0.4 and loaded into two factors were excluded . Structureviewed , 29.Cronbach’s alpha was used to evaluate the internal consistency for the total scale . The tesA total of 505 cancer patients completed the questionnaires, and five were subsequently excluded on account of considerable missing data (> 30%). Therefore, the effective response rate was 99%. The mean age of the participants was 58.03 years SD: 12.35), and 56.2% of the participants were females and 43.8% males. Approximately 76% of the patients were married/cohabiting. Furthermore, 76.2% of the patients had a high school level or lower educational level, and 82.8% were employed. Approximately 46.6% of the participants had a monthly income over 3000 yuan. The proportions of patients with lung cancer, breast cancer and haematological cancers were 20.8, 15.2 and 14.8%, respectively . However, the fit was poor, and 10 items were excluded due to low factor loading (< 0.5) and cross-loading on two factors. The remaining 29 items were analysed further, and 5 were extracted, explaining 57.21% of the variance . One factor (two items) was removed, as the explained variance of 4.59%  was lower than 5% [χ2 = 5382.19, df = 276, P < 0.001)  with a total explained variance of 56.26%.  [The goodness-of-fit of the four-factor structure of the PCQ-C tool was determined using AMOS version 17.0, which revealed excellent fit indices (χ= 0.951) –32. Accoα) for the total scale was 0.886, which is considerably over the recommended index of 0.70 [The Cronbach’s alpha coefficient , and Cronbach’s alpha coefficient (α) was adequate at both time points  since twenty patients rejected further investigation. The mean age of the participants was 57.04 years (SD: 12.32); 55% were females and 45% were males. The mean PsyCap-C scores at the first and second assessment occasions (T1 and T2) were 3.81 (SD = 0.51) and 3.64 (SD = 0.46), respectively. There was a significant correlation between the two periods  and anxiety .Among 227 patients with breast cancer, the mean scores of depression and anxiety were 6.83 (SD = 4.87) and 5.68 (SD = 4.23), respectively. PsyCap-C and the four individual dimensions showed a significant association with depression , and even relieve cancer-related fatigue –8, 33. HThe final version of the PCQ-C consists of four domains: hope (six items), self-efficacy (six items), resilience (six items) and optimism (six items). Hope is a positive expectation about achieving a possible and significantly good future in fighting cancer . Self-efOur study has developed the first objective measure of PsyCap specifically for cancer patients. We hope that it can be applied in the clinical setting to monitor PsyCap as part of cancer care and in psychological interventions as a self-report outcome measure.There were several limitations in the current study. First, the cross-sectional design precluded the verification of developmental stability or change in PsyCap for the sample. Therefore, the time-dependent changes in the sensitivity of the PCQ-C will have to be assessed in future studies. Second, although the PCQ-C was based on PsyCap theory, which mainly originated from studies in the West, we developed and validated this scale among Chinese cancer patients. Therefore, the applicability of the PCQ-C should be tested across different ethnicities. Third, our study lacked convergent validity for the scale and did not compare the PCQ-C with other scales. The self-reported data in our study might also have resulted in bias. Finally, items in the optimism dimension presented low item-total correlations compared to other items and will have to be revised.The PCQ-C is a reliable tool to objectively measure PsyCap among cancer patients based on self-efficacy, hope, resilience and optimism.


Patients from the SH- and WL-group who wanted further treatment after the 12 weeks were then offered the B4DT . There were no significant differences in symptoms of insomnia between the conditions at post-treatment, but a significant moderate improvement at 3-month follow-up for patients who received the B4DT. Insomnia was not associated with OCD-treatment outcome, and change in symptoms of insomnia was mainly related to changes in depressive symptoms. The main conclusion is that concentrated exposure treatment is effective irrespective of comorbid insomnia, and that insomnia problems are moderately reduced following treatment.Insomnia is a substantial problem in patients with obsessive-compulsive disorder (OCD). There is, however, a lack of studies investigating changes in concurrent symptoms of insomnia in OCD after concentrated treatment. A recent randomized controlled trial randomized participants to the Bergen 4-day treatment (B4DT,  Obsessive-compulsive disorder (OCD) is characterized by recurrent and anxiety provoking thoughts, images and/or impulses, that create the urge to perform compulsive behaviors or mental acts to reduce anxiety . LifetimGiven the extensive suffering many patients with OCD experience, little attention has been devoted to comorbid insomnia problems . InsomniComorbid sleep disturbances in OCD and anxiety disorders are not automatically resolved after treatment, and only moderate changes in sleep symptomatology can be expected . DespiteSince 2012, the OCD-team in Bergen, Norway, has developed a concentrated exposure-based treatment for OCD, the Bergen 4-day treatment (B4DT), where the treatment is delivered during four consecutive days. In a number of open trials it has been shown that 90% of the patients have responded at post-treatment assessment, and that nearly 70% are in remission at 3-month follow-up . FurtherIn a previous effectiveness study , we explThe current study is, to the best of our knowledge, the first controlled trial investigating changes in self-reported insomnia in patients with OCD. The aims of this study were: (1) To investigate differences in self-reported insomnia between patients who received B4DT with patients who received unguided self-help or were on a waitlist, (2) To investigate if insomnia is associated with treatment outcome for the primary OCD-disorder, (3) To investigate the relationship between depressive symptoms and insomnia. Based on our previous open trial , as wellClinicalTrials.gov (Identifier: NCT02886780). All participants provided written informed consent before inclusion in the study. For a full description of the study procedures, please refer to Launes et al. (n = 16), a 3-month unguided self-help  based on a manual by Foa and Kozak (n = 16). All patients were assessed the week prior to the intervention, as well as within a week after the intervention ended . The patients randomized to the B4DT were also assessed at 3-month follow-up.The current study is part of a randomized controlled trial that was carried out at Solvang, Sørlandet Hospital, in Norway, between August 2016 and September 2018. The study protocol was approved by the Regional Committee for Medical and Health Research Ethics of Western Norway, Bergen (REK Vest-2016/794) and was registered in s et al. . Eligiblnd Kozak , or a 3-Patients randomized to the SH- or WL-condition who wanted further treatment were offered the B4DT after the initial intervention. Twenty-six of these 32 patients (81.3%) started the B4DT after the initial intervention. Two patients had improved and four patients were not interested in further treatment. Therefore, a total of 42 patients eventually received the B4DT either as initial treatment or after SH or WL  or at 3-month follow-up .At post-treatment the treatment response rate was 93.8% in the immediate B4DT condition compared to 12.5% in SH, and 0% in WL . The ratAmong all participants in the RCT, there were 39 (81.3%) with indications of insomnia at pre-treatment. Of the 42 patients who were eventually treated with the B4DT, there were 35 (83.3%) patients with indications of insomnia at pre-treatment. For the three treatment conditions, there were three patients in each arm diagnosed with a sleep disorder. A summary of insomnia symptoms in the three conditions is displayed in 2(1) = 0.69, p = 0.406) or treatment condition (χ2(2) = 3.01, p = 0.222). Adding an interaction between time and condition resulted in an overall non-significant interaction effect, χ2(2) = 0.32, p = 0.852. Thus, there is no evidence that self-reported insomnia differed between patients who received B4DT and patients who received unguided self-help or were on a waitlist. There was a moderate reduction in BIS scores at follow-up for the immediate B4DT group (d = 0.43). See An HLM with time and treatment condition as independent variables revealed no statistically significant effects of time (χn = 42), a separate HLM was conducted on the subsample who had indication of insomnia pre-treatment according to BIS . This subsample included patients who had received B4DT either as first treatment (n = 16) or after first participating in SH (n = 13) or WL (n = 13). The analysis showed a statistically significant overall effect of time, χ2(2) = 26.22, p < 0.001, with a significant decrease in BIS scores from pre-treatment to 3-month follow-up, b = −4.79, 95% CI = −7.35, −2.24, p < 0.001, but not from pre-treatment to post-treatment, b = 0.54, 95% CI = −1.96, 3.04, p = 1.00.To investigate change in self-reported insomnia for all patients who eventually received the B4DT (n = 16) or after first participating in SH (n = 13) or WL (n = 13). The results revealed an overall statistically significant effect of time, χ2(2) = 504.21, p < 0.001. The patients scored significantly lower at both post-treatment  and follow-up  compared with pre-treatment. Including an interaction between time and insomnia (patients with vs. patients without indications of insomnia at pre-treatment) resulted in a statistically significant interaction effect, χ2(2) = 6.27, p = 0.044. The results showed that patients with insomnia at pre-treatment had a steeper decrease from pre-treatment to follow-up than patients without insomnia, b = −5.64, χ2(2) = 6.25, p = 0.044. There was no statistically significant difference from pre-treatment to post-treatment between the insomnia and non-insomnia groups, χ2(2) = 1.89, p = 0.169.A comparison between the insomnia and non-insomnia groups is presented in 2(2) = 2.90, p = 0.235. The results showed that pre-treatment BIS did not have a statistically significant effect on Y-BOCS scores at post-treatment  or follow-up .This analysis was repeated using continuous BIS scores instead of a dichotomous (insomnia vs. non-insomnia) variable. Including an interaction between time and BIS resulted in a statistically non-significant interaction effect, χd = 1.14) than the non-insomnia group (d = 0.51). A HLM revealed that both the within-person variation and the between-person variation in depressive symptoms had a statistically significant association with BIS scores. Patients with above average depressive symptoms on average also scored higher on BIS across the three measurement points . Additionally, those patients who at one time-point exhibited an increase in BIS scores  also tended to report an increase in BIS scores .There were significant changes in depression following treatment. This change (pre-treatment to follow-up) was more evident for the insomnia group  was To the best of our knowledge, this is the first study to investigate changes in self-reported symptoms of insomnia in patients with OCD undergoing treatment in a randomized controlled trial. This topic is clinically relevant as previous studies have shown that symptoms of insomnia are present in 50–80% of all patients with a psychiatric disorder , 36. At Although 81% had indication of insomnia, only 19% met DSM-5 criteria for a sleep disorder. This discrepancy is likely due to differences between DSM-IV and DSM-5 criteria and differences when using self-reported symptoms and diagnostic assessors. The BIS was based on DSM-IV-TR where symptoms only had to last 1 month. In comparison, the DSM-5 criterion is 3 months. Furthermore, indications of insomnia is not the same as diagnosis, but the BIS can be used to screen for insomnia.There were no statistically significant differences in BIS scores between the three conditions at pre- or post-treatment, but patients in the B4DT condition showed improved sleep at follow-up. This suggests that even though the B4DT is effective in reducing primary OCD symptoms and secondary symptoms  at post-treatment, symptoms of insomnia did not show a similar immediate improvement. The lack of significant findings post-treatment is most likely due to the brief treatment format and the fact that the BIS measure assesses sleep problems occurring during the past month. Since the B4DT only lasts for 4 days, it would be unrealistic to expect significant differences post-treatment. Another possible explanation might be that symptoms of insomnia alleviate over longer periods of time (than 4 days) as the patients are continuously working with the exposure principles and treatment plan , 39. Whid = 0.53). A possible explanation is that the B4DT has a generic effect as the B4DT is not only effective in treating the principal symptoms, but also has a positive effect on comorbid symptoms. This finding is in accordance with a meta-analysis by Belleville et al.  was also particularly high in the current sample. Future studies should consider to investigate long-term follow-up outcome to further explore residual symptoms of insomnia in OCD and how this might affect treatment response and symptom recurrence . This coIn conclusion, the results indicate that symptoms of insomnia in OCD-patients do not impair the primary treatment outcome, and is moderately improved after specific treatment. However, residual insomnia symptoms should be expected and conceivably be dealt with to prevent relapse and recurrence in a follow-up period.kristen.hagen@helse-mr.no.The datasets generated for this study are available on request to the corresponding author. Requests to access these datasets should be directed to ClinicalTrials.gov (Identifier: NCT02886780). The patients/participants provided their written informed consent to participate in this study.The trial received ethical approval from the Regional Committee for Research Ethics  and was registered in GK, L-GÖ, BH, KH, and GL contributed to the study design. GL and KH contributed to the data collection. KH, HN and SS drafted the manuscript. KH, SS and SH conducted the statistical analysis. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coeliac disease (CD) patients are distinguishable from healthy individuals via urinary volatile organic compounds (VOCs) analysis. We exposed 20 stable CD patients on gluten-free diet (GFDs) to a 14-day, 3 g/day gluten challenge (GCh), and assessed urinary VOC changes. A control cohort of 20 patients continued on GFD. Urine samples from Days 0, 7, 14, 28 and 56 were analysed using Lonestar FAIMS and Markes Gas Chromatography–Time of Flight–Mass Spectrometer (GC-TOF-MS). VOC signatures on D (day) 7–56 were compared with D0. Statistical analysis was performed using R. In GCh patients, FAIMS revealed significant VOC differences for all time points compared to D0. GC-TOF-MS revealed significant changes at D7 and D14 only. In control samples, FAIMS revealed significant differences at D7 only. GC-TOF-MS detected no significant differences. Chemical analysis via GC-MS-TOF revealed 12 chemicals with significantly altered intensities at D7 vs. D0 for GCh patients. The alterations persisted for six chemicals at D14 and one (N-methyltaurine) remained altered after D14. This low-dose, short-duration challenge was well tolerated. FAIMS and GC-TOF-MS detected VOC signature changes in CD patients when undergoing a minimal GCh. These findings suggest urinary VOCs could have a role in monitoring dietary compliance in CD patients. Coeliac disease (CD) is a T-cell mediated gluten sensitive enteropathy, affecting approximately 1% of the global population ,3. The vTissue sampling for diagnosis of CD requires the consumption of a gluten-inclusive diet for at least 8 weeks prior to biopsy  demonstrThe detection of specific patterns of volatile organic compounds (VOCs) using multiple technologies, including electronic noses (E-noses) and related technology, in urine, breath, sweat and faeces has been applied to the non-invasive diagnosis of gastrointestinal conditions, including inflammatory bowel disease, bile acid malabsorption, colorectal cancer and radiation-induced GI side effects ,15,16,17In an earlier study we used field asymmetric ion mobility spectrometry (FAIMS) to distinguish CD patients from diarrhoea predominant irritable bowel syndrome (D-IBS) via urinary VOC analysis with a sensitivity and specificity of 85% . FAIMS aA study of faecal VOCs in refractory CD patients and CD patients on a GFD using gas chromatography–ion mobility spectrometry (GC-IMS) technology, found that refractory CD could be differentiated from CD on a GFD with an area under the curve (AUC) of 0.91 . These sOur study aimed to analyse urinary VOCs in stable CD patients on a GFD undergoing a 14-day GCh of biscuits containing 3 g of gluten/day. The overall purpose of these experiments was to determine whether the urinary VOC signatures of stable CD patients changed in response to GCh, and whether it reverted to the pre-challenge signatures after the cessation of the challenge. To achieve this, we used a combination of a FAIMS technique (E-nose family), which can be deployed as a point of care test and has previously shown applicability to inflammatory conditions, with GC-TOF-MS (gas chromatography-time of flight-mass spectrometry) to identify specific biomarkers resulting from the challenge, and to determine whether the two technologies gave comparable results.n = 20) or to continue with their GFD . The non-GCh patients acted as a control group.Forty stable, long-term CD patients, diagnosis confirmed histologically, with self-reported compliance with GFD and a normal TTG in the preceding 3 months, were recruited. They were asked to either receive a gluten challenge , daily in addition to their regular GFD for 14 days. Each cracker biscuit contains 1 g of gluten  so each Urine samples were collected from both groups on day 0 (pre-GCh), and on days 7 and 14 (end of GCh), and then on days 28 and 56. Five millilitre blood samples for serum TTG concentration were also collected on Days 0, 14 and 56. Patients completed a symptom diary and were allowed to decrease or stop the GCh if symptoms became intolerable. Analyses were done on an intention-to-treat basis.Patients provided 20 mL samples of early morning urine, which were voided into standard universal Sterilin specimen containers  and stored at −80 °C within 24 h of voiding. Urinary VOCs have been shown to be stable at room temperature during this period . These sFAIMS is an approach where VOCs can be detected by measuring their mobilities in high and low electric fields. Molecules are ionized and then passed between two plates to which an asynchronous voltage is applied. This alters the path of the ion, which is compensated for by applying a DC voltage and allowing the ion to be detected. Thus, the mobility measurement is a function of the amplitude of the high/low electric fields and the compensation voltage.For the FAIMS analysis, a commercial instrument was used . This was attached to an ATLAS sampling system . In use, a 5 mL sample aliquot was placed into a 10 mL vial. The sample was then placed in the ATLAS sampling system and heated to 40 ± 0.1 °C for 10 min. The flow rate over the sample was set to 200 mL/min using clean/dry air as the carrier gas. The sample air was further diluted with zero air to create a total flow of 2 L/min. The Lonestar was scanned from 0 to 99% dispersion field in 51 steps and +6 V to −6 V compensation voltage in 512 steps, with both positive and negative ions used. This created a output file per sample of 52,224 data points. Each sample was scanned three times and air blanks were used in between samples to ensure that there was no carry over. Furthermore, our standard quality control checks were performed before starting the experiment to ensure the results were reliable.GC-TOF-MS is a hybrid technology composed of a gas chromatography column in sequence with a time-of-flight mass spectrometer. This allows the separation of chemical compounds before they are applied to the mass spectrometer and provides better separation for analysis.For GC-TOF-MS, 5 mL of defrosted urine sample was aliquotted into a 20 mL borosilicate vial  containing a Markes Bio-Monitoring sorbent tube , and sealed with a crimp cap. The samples then heated for 60 min at 40 °C to absorb the urine onto the sorbent tube for analysis.Once complete, the sorbent tubes were sealed using Markes DiffLok caps and loaded into a Markes Unity and Ultra Thermal Desorber auto-sampler system for analysis using a Markes BenchTOF-HD using a Rxi-624SilMS 20 m 0.18 mm ID, 1.0 um column (Thames Restek 13865).The TOF-HD analysed masses between 35–250 atu (atomic mass units) for a total run time of 25 min. The filament voltage was set to 1.7 V, and the filament delay to 10 s. The sample transfer line was set to 250 °C, as was the ion source temperature, with ionisation at −70 V. During the analysis dynamic background compensation (DBS) was automatically applied by the software, which integrates and deconvolutes the peaks present in the sample. The integration settings were as follows: global height reject, 10,000; global width reject, 0.001; baseline threshold, 3; and global area reject, 10,000. The peaks were then identified using the NIST list, with a forward and reverse match with a minimum match factor of 450. These settings were used based on previous clinical studies involving urine samples  In both Blood samples were collected in standard 5 mL biochemistry bottles and then analysed using the EliA Celikey IgA assay , as per the manufacturer’s guidelines. The normal range of the assay is <5 kU/L.Although no formal calculation of sample size was performed, previous VOC studies performed by our group have shown that sample and control groups of 15–20 patients are able to achieve statistically significant results ,17,18.p-value were then used to create a model. A sparse logistic regression model was applied from the resultant probabilities; statistical parameters including ROC (receiver operator characteristics) curves, sensitivity, and specificity, area under the curve, negative predictive value, positive predictive value and p-value were calculated.All statistical analyses were performed using R version 3.4.1. For the Lonestar analysis, all the samples were exported to text file and the second run used for analysis. From previous studies, we have found that the second run provides the most reliable information content. The positive and negative scans were combined into a single matrix and a threshold applied to remove the background information. In this case, any values below the threshold were set to zero and were set at twice the average background level, with the same value used for all the files. Once completed, a number of comparisons were undertaken comparing different days to day 0. For each comparison, a 10-fold cross validation was used, where the data are divided into 10 equal groups, with 9 groups using to create a model that was applied to the 10th group. This was repeated 10 times until all the samples were within a test group. For the training group, a rank-sum test was performed on each data point, across all the samples, to identify those that held discriminatory information. The 20 data points with the lowest T-test analysis with a p value of <0.05 to identify significantly different chemicals between each time point.For the GC-TOF-MS, a similar 10-fold cross validation process was undertaken. Here, the identified chemicals were used as features and the abundance as its value. In addition, due to the smaller dimensionality of the data at this point, no feature extraction approach was required and the models were built directly on the output values. Analysis of the chemical composition and differences between each time point and Day 0 was performed using a paired There were no significant differences in age and sex when the GCh and non-GCh patients were compared. However, one of 20 GCh patients returned no samples. Of the remaining 19 patients, 18 ingested the crackers as per protocol and one patient reduced their crackers intake in the last 3 days by 25%. This patient was included in the analyses. These 19 patients returned their urine samples and symptom diaries.Three of 20 non-GCh patients returned no samples and 17 of 20 returned their urine samples and symptom diaries. The pre-GCh urine sample signatures on Day 0 were compared to the signatures of the samples on Days 7, 14, 28 and 56 using the sparse logistic regression model.n = 19), when compared to Day 0, the signatures were significantly different on days 7, 14, 28 and 56. The AUC decreased from 0.92 (0.85–0.99) on Day 7, to 0.80 (0.64–0.95) on Day 14, to 0.71 (0.53–0.89) on Day 28 and 0.77 (0.62–0.93) on Day 56. P values for the four time points were <0.001, <0.001, 0.01 and 0.002, respectively.Among the GCh patients (p = 0.002), but not on Days 14, 28 and 56.Among the non-GCh patients, when compared to Day 0, there was a significant difference in VOC signatures on Day 7 , compared to Day 0, signatures were significantly different on Days 7 and 14. When comparing Day 0 with Day 7 and Day 0 with Day 14, the sensitivities were 0.65 (range 0.44–0.83) and 0.71 (range 0.52–0.88), the specificities were 0.64 (range 0.42–0.85) and 0.69 (range 0.45–0.90) and the p values were 0.02 and 0.05, respectively. There was no difference in VOC signatures when Days 28 and 56 were compared to day 0.Among the GCh patients , remained altered until Day 56. Among the non-GCh patients, no consistent chemical alterations was detected throughout the study period.p values from the analysis.On review of the symptom diaries for the GCh patients, 73.5% (14/19) reported bloating, lethargy, loose stools and diarrhea that persisted during the challenge period. These symptoms were not sufficiently disabling to cause a reduction in gluten consumption for 18 out of 19 subjects; none terminated the challenge. Their symptoms resolved within a mean of 4.1 (range of 0–41) days post-challenge.The non-GCh patients reported fewer symptoms than the GCh patients, with 53.0% (9/17) reporting brief episodes of bloating or loose stools; none for more than 24 h duration, and none coinciding with the significant VOC difference seen on Day 7.Among the GCh patients, mean TTG antibody levels were 2.43 U/mL, 3.6 U/mL and 3.58 U/mL, at Days 0, 14 and 56, respectively. The increase was non-significant. In the non-GCh patients, the mean TTG antibody was unchanged at 1.17 U/mL, 1.20 U/mL and 1.10 U/mL on Days 0, 14 and 56, respectively. These results are shown in The ingestion of cracker biscuits was associated with increased gastrointestinal symptoms among the GCh patients, although only 1 of 19 patients reduced their cracker biscuit intake, and only for the last 3 days of the challenge. There were only mild, brief non-specific symptoms among the non-GC patients, suggesting that the GCh patients’ symptoms could be attributed to the ingested gluten.We used FAIMS and GC-TOF-MS to analyse identical urine samples and see if changes could be detected by both methods to lend authenticity to the observations. The GC-TOF-MS analysis allowed the identification of specific chemicals.Both techniques identified a change in VOC signatures on Days 7 and 14 among patients who underwent the challenge. This change was no longer apparent by Day 28 when assessed by GC-TOF-MS, but was still detectable up to the final study time point (Day 56) by FAIMS analysis. There was no accompanying significant increase in TTG antibodies during the challenge, suggesting that VOC signature changes occur prior to any changes in this biochemical marker. If this is due to gluten, the presence of changes in VOCs by day 7 of the challenge suggests that this method of assessment is highly sensitive to gluten ingestion in stable CD patients. This could indicate a potential role for the technology in non-invasive CD monitoring and follows an earlier study to demonstrate faecal VOC changes in refractory CD, compared to those in remission on GFDs .Urine samples were snap-frozen on receipt of samples, but previous studies suggest VOCs can still be assessed for up to 72 h after voiding  at room The underlying reasons behind why ingestion of three crackers biscuits daily might alter VOCs signatures in patients with coeliac disease stable on a GFD is uncertain. It could be a non-specific effect unrelated to the small amount of gluten contained in these biscuits. However, the accompanying symptomatic response in the GCh patients supports the view that gluten could be the cause of the changes noted in VOC signatures in urine.FAIMS method detected ongoing VOC alterations after the GC ended, up until Day 56. The strength of signal decreases as shown by the decreasing AUC, sensitivity and specificity. FAIMS might detect changes caused by gluten ingestion that are more persistent. In contrast, the GC-TOF-MS methodology seems to detect changes occurring during the period when gluten is still being ingested. The underlying mechanism behind these changes could be an alteration of the GI microbiome or small bowel duodenal inflammation caused by gluten, although the latter is more plausible given the promptness of the changes noted. An increase in small intestinal permeability associated with gluten-induced inflammation might lead to luminal substances entering the circulation and subsequently excreted in urine. The discrepancy between the two methodologies could be that while the FAIMS detects the effects of gluten-induced mucosal inflammation, the GC-TOF-MS detects a component of ingested gluten. Gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) hybrid technology is able to detect masses between 35–250 atomic mass units, but is novel in regard to its intended purpose in this study. Previous studies have used field asymmetric ion mobility spectrometry (FAIMS) to distinguish between patients with refractory CD from those that respond to GFD.Chemical analysis using the GC-TOF-MS VOC assay showed among the GC patients that the abundance of 12 chemicals decreased by an average of 52% by Day 7. Only six chemicals were still decreased on Day 14. One chemical, N-methyltaurine, was decreased at all time points. N-Methyltaurine is an organosulphonic acid and has been previously identified as a common untargeted urinary metabolite . It is uHalf of the observed chemical changes had normalised by the end of the GCh, with N-methyltaurine still abnormal at Day 28. By Day 56, there were no residual chemical changes. These changes may represent either the presence of gluten in the diet or alterations in the patients’ metabolic processes induced by small intestinal inflammation and/or alterations in the gut microbiome. Further studies with the corresponding microbiome analysis and small intestinal histological sampling would be needed to fully elucidate the underlying processes involved.In non-GC patients, urinary VOCs remained unchanged for the duration of the study using GC-TOF-MS and were significantly different for one time point using FAIMS; this may represent background dietary gluten contamination. Future studies could consider the use of urinary gluten immunogenic peptide (GIP) to determine whether accidental gluten exposure has taken place in the control cohorts.There are a number of limitations of this study. First, the FAIMS system and the GC-TOF-MS may not detect the same chemicals. The FAIMS system is undertaking direct headspace sampling, whilst the GC-TOF-MS process uses a pre-concentration stage. The tube used on the GC-TOF-MS was designed for measuring chemicals C4 and above; therefore, chemicals below C4 are unlikely to be detected. In addition, it was not possible to identify the specific chemicals detected by the FAIMS system, but the improved results for FAIMS may indicate that the chemicals being detected have a high proton affinity or are smaller and were not collected onto the tube. The advantages of FAIMS are that the technology is cheaper than GC-TOF-MS and that it can be used away from a lab setting and does not require specialised gases to operate. This makes it a potentially clinically useful approach in the future. A second limitation is that we did not undertake chemical confirmation of those identified in this study nor performed calibration for concentration. However, this is common with pilot studies of this nature and, if such a test is to be used in the future, further work will be necessary to confirm and quantify these chemicals.Further limitations related to study design include the levels of gluten consumed for the challenge. Whilst sufficient to induce symptoms in our cohort, and equivalent doses have previously shown histological changes in the small bowel , it is wThis study provides further evidence of the potential of a role for urinary VOC analysis in patients with CD, and demonstrates the first use of GC-TOF-MS for this purpose. Earlier studies have demonstrated that FAIMS could distinguish CD from healthy controls and D-IBS. Here, we demonstrate that a 14-day GCh results in alterations in VOC signature changes in the urine of stable CD patients using both FAIMS and GC-TOF-MS. These changes appeared by Day 7 and resolved after completion of the GCh as shown by GC-TOF-MS technology. Our findings suggest a role for urinary VOC assessment in monitoring GFD compliance in CD patients where the inadvertent ingestion of small amounts of gluten can be a problem.